CRYSTAL GROWTH & DESIGN
Effect of Omega-3 Fatty Acids on Crystallization, Polymorphic Transformation and Stability of Tripalmitin Solid Lipid Nanoparticle Suspensions Tarek Samir Awad,*,† Thrandur Helgason,‡,§ Jochen Weiss,‡ Eric Andrew Decker,† and David Julian McClements†
2009 VOL. 9, NO. 8 3405–3411
Department of Food Science, UniVersity of Massachusetts, Amherst, Massachusetts 01003, Department of Food Science and Biotechnology, UniVersity of Hohenheim, Garbenstr. 25, 70593 Stuttgart, Germany, and Department of Food Science and Nutrition, UniVersity of Iceland, Hjardarhagi 2-6, ReykjaVik, 107, Iceland ReceiVed October 17, 2008; ReVised Manuscript ReceiVed June 3, 2009
ABSTRACT: We examined the effect of lipid phase composition on the crystallization, polymorphic transition and stability of solid lipid nanoparticle (SLN) suspensions. A series of fine-disperse oil-in-water emulsions was prepared at an elevated temperature (75 °C) from a lipid phase containing different amounts of a high melting lipid (tripalmitin) and a low melting lipid (fish oil). These emulsions were cooled to induce crystallization and form SLN suspensions. In the absence of fish oil, the SLN suspensions formed a gel after the emulsified tripalmitin crystallized, which was attributed to particle shape changes leading to aggregation and network formation. Light scattering and rheology measurements indicated that incorporation of fish oil into the lipid phase (g10 wt %) increased the stability of SLN to aggregation. DSC measurements suggested that crystallization, melting, and polymorphic transitions of SLN were influenced by the amount of fish oil incorporated. The rate of R- to β-polymorphic transitions of tripalmitin increased with increasing fish oil content, and tripalmitin crystals formed appeared to be less ordered as evidence by a lower melting temperature. Results suggest that the phase behavior and morphology of tripalmitin crystals can be altered by mixing them with low melting lipids such as fish oil thereby improving the stability of SLN suspensions to particle aggregation and gelation. Moreover, results show that fish oil, rich in ω-3 polyunsaturated fatty acids, can be successfully incorporated into SLN suspensions. Introduction There is growing evidence that diets rich in omega-3 polyunsaturated fatty acids may aid in the prevention of coronary heart diseases (CHD)1-4 and cerebrovascular accidents (CVA)5 resulting in decreases in associated mortalities.6 However, the general populations of many developed countries currently consume levels of omega-3 polyunsaturated fatty acids that are well below those believed to be required to be beneficial to consumers’ health. Consequently, there is a major thrust within the food industry to develop functional foods that are fortified with omega-3 fatty acids, such as fish, flaxseed and algae oils. A major challenge to fortifying foods with omega-3 fatty acids is that they are highly susceptible to lipid oxidation, leading to rancid off-flavors and potentially toxic reaction products. Effective strategies therefore need to be developed to encapsulate and protect omega-3 fatty acids in functional food products against oxidation during production, packaging, storage, transport and retailing. Solid lipid nanoparticle (SLN) suspensions are an important class of emulsion-based delivery systems with a number of potentially beneficial attributes for encapsulating omega-3 fatty acids, such as high loading capacity, good chemical and physical stability, good oral bioavailability, and potential for large scale production.7-11 SLN have proven to be a powerful means of delivering poorly water-soluble drugs in the pharmaceutical industry.7,11,12 They may thus also have potential to protect and deliver lipophilic functional agents in the food industry.13-15 SLN suspensions consist of fully or partially crystalline lipid * To whom correspondence should be addressed. E-mail:
[email protected]. Phone: (1) 413-687-0770. Fax: (1) 413-545-1262. † University of Massachusetts. ‡ University of Hohenheim. § University of Iceland.
nanoparticles suspended in an aqueous continuous phase.8,10,16,17 The stability of SLN suspensions to aggregation, as well as their ability to retain and protect encapsulated components during storage, is strongly dependent on the physical state of the lipids.13,14,18-21 Previous studies in our laboratories have shown that SLN suspensions are highly susceptible to particle aggregation and gel formation, via a mechanistic pathway that is related to R- to β-polymorphic transitions in triacylglycerides used to form the base matrix of the nanoparticles.13,14 This transition may destabilize SLN particles both physically and chemically, which decreases their potential use for the encapsulation of sensitive bioactive ingredients, such as omega-3 fatty acids. Recent studies have shown that cooling and heating rates, holding time and temperature, and surfactant type greatly influence the rate of polymorphic transformations and the incidence of gelation.13,14,22 In this study, we examined the impact of incorporating fish oil into tripalmitin SLN on their physicochemical properties and stability. Previous studies have shown that mixing low melting point lipids (such as fish oils) with high melting point lipids (such as tripalmitin) governs the overall phase behavior of the mixed system, e.g., melting, crystallization and polymorphic transitions.23,24 Ultimately, we want to determine whether stable SLN containing fish oils can be produced, since these systems could then be used to encapsulate, stabilize and deliver omega-3 fatty acids. Materials and Methods Materials. Tripalmitin was purchased from Fluka (Buchs, Switzerland). Sodium phosphate monobasic and sodium phosphate dibasic were purchased from Fisher Scientific (St. Clair Shores, MI). Sodium azide and polyethylene glycol sorbitan monolaurate (Tween 20) were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO). Menhaden oil (eicosapentaenoic acid (EPA), 12-15%; docosahexaenoic
10.1021/cg8011684 CCC: $40.75 2009 American Chemical Society Published on Web 06/19/2009
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acid (DHA), 10-14%; total ω-3 PUFAS, 25-27%) obtained from Omega Protein, Inc. (Reedville, VA) was stored at -80 °C upon arrival and thawed in cold tap water 15 min prior to use. All chemicals were used as received. Methods. (a) Solid Lipid Nanoparticle Preparation. Lipid phases were prepared by mixing fish oil and tripalmitin at a temperature of 75-80 °C above the melting point of tripalmitin. An aqueous phase was prepared by mixing a water-soluble surfactant (1.5% (w/w) Tween 20) with buffer solution (4 mM sodium phosphate monobasic, 6 mM sodium phosphate dibasic, 0.02% sodium azide, pH 7) and then heating the solution to 75 °C. 10 wt % oil-in-water emulsions were prepared by first blending the lipid and aqueous phases together at 75-80 °C for 1 min using a high speed blender (model SDT-1810, EN Shaft, Tekmar Co., Cincinnati, OH) and then passing them through a high pressure homogenizer (Microfluidics, Newton, MA) 10 times at 9 kbar. This procedure produced emulsions with a monomodal size distribution and a mean particle diameter of around 145 nm. The emulsions were stored in plastic containers at 37 °C in a temperature-controlled room prior to analyses. No change in mean particle diameter was observed after 2 months of storage, and the lipid phase did not crystallize, indicating that these emulsions contained liquid droplets that were stable to aggregation. These emulsions were used to form SLN by cooling them into an ice bath. (b) Particle Size Determination. Particle size measurements were performed by photon correlation spectroscopy (PCS) using a Malvern Zetasizer NanoZS (Malvern instruments, Malvern, U.K.). The samples were diluted 100× in the buffer solution at 37 °C. PCS gives the mean diameter of the particle population and the polydispersity index (PI) ranging from 0 (monodisperse) to 0.50 (very broad distribution).25 To monitor the initial emulsion stability, the particle size distribution was measured at 37 °C soon after preparation and during a storage period of 4 months. At this temperature the lipid phase of the emulsions remained liquid. To monitor the aggregation and shape change of the particles, which could be induced by lipid crystallization and polymorphic transformations,13,14 we measured the hydrodynamic diameter of the nanoparticles after the formation of SLN at 10 °C for several hours. (c) Solid Fat Content (SFC). The solid fat content was determined using a low-resolution NMR spectrometer Minispec MQ20 (Bruker Optics GmbH, Rheinstetten, Germany) with a mq-PHH20-10-25/R/V probe, operating at 19.5 MHz proton frequency. The free induction decay (FID) was recorded after a 90°-pulse excitation (SPE, single pulse excitation), i.e. 90°x-dead time-acquisition of the amplitude A(t) of the transverse magnetization as a function of time t. The duration of a 90° pulse was 2.26 µs, the dead time was 7 µs. Tripalmitin/fish oil mixtures were melted at 85 °C for 30 min to ensure complete melting, and then mixed using a vortex to ensure homogeneity of the samples. Glass NMR tubes (10 mm diameter) were filled with approximately 3 g of each sample and kept in a refrigerator at 5 °C for 24 h. SFC (%) measurements for each mixture were then taken in triplicate. (d) Differential Scanning Calorimetry (DSC). A differential scanning calorimeter (DSC; Q1000, TA Instruments, New Castle, DE) was used to study crystallization, melting and polymorphic behavior of the different formulations during several cool-heat cycles at constant cooling and heating rates. An aliquot of emulsion (8-10 mg) was placed in a hermetic aluminum pan and sealed. An empty pan was used as a reference. All the DSC pans were preheated to 80 °C before adding the sample to avoid crystallization and/or phase separation of the lipid mixture. Constant heating (10 °C/min) and cooling (5 °C/min) rates were used to study the phase transitions of emulsions containing different ratios of fish oil and tripalmitin. (e) Rheology. Storage modulus (G′) and loss modulus (G′′) of emulsions undergoing sol-to-gel transitions were measured using an oscillatory rheometer (Paar Physica MCR 300, Anton Paar, Graz, Austria) using a cone and plate measurement system (diameter 49.94 mm, angle 2°) thermostatted by a Peltier system. The cone was positioned 50 µm above the plate to avoid artifacts arising from the presence of any large particles in the samples. Initially, the linear viscoelastic range was determined by conducting a strain sweep at an oscillation frequency of 1 Hz. The strain sweep indicated that G′ and G′′ of gelled samples did not decrease at strains less than 0.8%, and consequently all gelation experiments were conducted at a constant strain of 0.1% and frequency of 1 Hz. The temperature of the loaded sample was equilibrated to 37 °C using the Peltier system before any cooling/heating run. After loading the nanoemulsion sample at 37 °C,
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Figure 1. DSC exotherms (up) and endotherms (down) of bulk tripalmitin (a) and 75% tripalmitin/25% fish oil mixture (b) during a cool-heat cycle. R, β′ and β are the three polymorphic forms; Tc and Tm are crystallization and melting temperatures; R1 and R2 are β′ and β recrystallization peaks. it was cooled down to 5 °C at a constant cooling rate of 5 °C/min. Since the polymorphic transformation that may induce gelation may occur during the heating process (i.e., via melt-mediation), the samples were subsequently heated from 5° to 75 °C at a constant heating rate (5 °C/min). (f) Visual Observation. Sample fluidity and the incidence of gelation were also detected by visual observation. Emulsion samples stored at 37 °C were transferred into glass tubes, which were then stored in a refrigerator at 5 °C for 1 day. The glass tubes were then inverted to determine whether they remained fluid (flowed to the bottom) or became solid-like (stayed at the top). To investigate the occurrence of particle coalescence during the cool-heat cycle, the samples were heated from 5 to 75 °C and the degree of any oil separation was observed visually.
Results and Discussion Effect of Fish Oil on Crystallization of Bulk Tripalmitin. The crystallization and melting behavior of a series of bulk tripalmitin/fish oil mixtures was measured using DSC (Figure 1). When 100% tripalmitin was cooled from 75 °C, a sharp exothermic peak was observed around 39 °C (Figure 1a), which can be attributed to its crystallization into the metastable R-form.26 Upon heating, a series of endothermic and exothermic peaks were observed in the thermogram for the pure tripalmitin system, which can be assigned to particular lipid phase transitions.26 The endothermic peak at 41 °C corresponds to melting of the R-form; the exothermic peak at 47 °C corresponds to recrystallization of the R melt into the more stable β′-form; the small endothermic peak at 53 °C corresponds to melting of the β′-form; the exothermic peak at 56 °C corresponds to recrystallization of the R melt into the most stable β-form; and the large endothermic peak at 63 °C corresponds to melting of the β-form.
Stability of Omega-3 Encapsulated SLN Suspensions
Figure 2. (a) DSC crystallization exotherms of bulk tripalmitin containing various concentrations of fish oil (0-100%) during cooling from 80 to 5 °C and (b) plot of crystallization temperature (Tc) versus percentage of the fish oil (Φ).
The addition of a relatively small amount of low melting lipid (fish oil) to the lipid phase caused an appreciable alteration in its thermal behavior (Figure 1b). When 25% fish oil/75% tripalmitin was cooled, a single crystallization peak was still observed, but it occurred at a slightly lower temperature (35 °C) than for the 100% tripalmitin (39 °C). During the subsequent heating process only two peaks were detected: a small broad exothermic peak at 34 °C, which can be assigned to a meltmediated polymorphic transition from the metastable R- to β-form; and, a large broad endothermic peak at 64 °C, which can be attributed to melting of the β-form. The incorporation of fish oil into the lipid phase therefore had a pronounced influence on the thermal behavior of tripalmitin. We then examined the impact of fish oil concentration on the thermal behavior of the bulk tripalmitin (Figures 2 and 3). The peaks observed in the cooling and heating thermograms varied in position, shape, and magnitude depending on the lipid composition. First, the crystallization temperature (Tc) shifted to lower temperatures with increasing fish oil content, e.g., from 39 to 27 °C for 0 to 75% fish oil (Figure 2a). No crystallization peak was observed at 100% fish oil, since fish oil did not crystallize in the temperature range studied. Therefore, increasing the liquid oil mass fraction caused a linear decrease in Tc (Figure 2a) as expected from colligative property arguments (freezing point depression).23,24 Second, a broadening in the crystallization peak was observed with increasing fish oil content from 0 to 25%, while two peaks were observed at higher fish oil contents. For example, for the 50% fish oil mixture there was a large narrow peak at 33 °C and a smaller broad peak at about 27 °C. The broadening and splitting in the peaks may be explained by a solid state polymorphic transformation from the metastable R-form to a more stable form during the cooling step. Presumably, the presence of the unsaturated triacylglycerol
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Figure 3. (a) DSC melting endotherms of bulk tripalmitin containing various concentrations of fish oil (0-100%) during heating from 5 to 80 °C and (b) plot of the β melting temperature (Tm) versus percentage of the fish oil (Φ).
molecules in the liquid fish oil increased the mobility of the saturated tripalmitin molecules thereby enabling them to reorganize their crystal morphology more rapidly. The melting thermograms of crystallized bulk tripalmitin containing different amounts of fish oil are shown in Figure 3a. The addition of 5% fish oil to the tripalmitin caused a distinct change in the melting behavior of the mixed lipid phase, which can be attributed to the fact that a portion of the R-phase was converted to β-phase during the cooling process (Figure 2a). From 10 to 75% fish oil, only a single endothermic peak was observed. This peak appeared at a relatively high temperature and can therefore be assigned to the melting of the stable β-form, again confirming that the majority of the R-phase was converted into the β-phase during the cooling process. The melting temperature (Tm) of the β-form of tripalmitin decreased as the fish oil content increased (Figure 3b), which suggests that the tripalmitin crystals did not pack as efficiently in the presence of fish oil. The DSC results indicated that, with increasing the ratio of liquid oil, the melting temperature decreased and that the melting peak broadened and became less symmetrical. This can be explained by the solubility effect of the fish oil, which increases with increasing the fish oil/high melting fat ratio.27 The decrease in the melting point (Figure 3b) at high fish oil content may thus be indicative of increasing solubility. The solubility of tripalmitin in fish oil was studied using the Hildebrand solubility model.28,29
log10 x )
(
∆H 1 1 R T HMF Tm m
)
(1)
where x is the mole fraction of solid fat (x ) SFC/100), R is the universal gas constant (8.314 J/(mol K)), ∆H the enthalpy of the mixture melt (in J/mol), and TmHMF and Tm are the peak
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Figure 4. (a) Solid fat content (SFC) as a function of fish oil ratio (Φ), and (b) Hildebrand plot of tripalmitin containing various concentrations of fish oil.
melting temperatures of pure tripalmitin and the tripalmitin/ fish oil mixture (in K), respectively. A straight line in a log10 x vs 1/Tm plot is indicative of ideal solubility of the tripalmitin crystals in the fish oil. Figure 4a shows the SFC values as a function of the fish oil ratio in the mixture. As can be seen, increasing the amount of fish oil decreased the SFC in a linear fashion. Figure 4b shows the plot of log10 x vs 1/Tm for tripalmitin/FO mixture, which clearly demonstrates a linear relationship between the two parameters, indicating that tripalmitin formed an ideal solution in the fish oil. The heat of fusion of pure tripalmitin determined from the slope of the solubility curve was 243 kJ/mol, which is close to that measured experimentally (234 kJ/mol). Effect of Fish Oil on Crystallization of Emulsified Tripalmitin. DSC thermograms of O/W emulsions (d ∼ 145 nm) containing different ratios of fish oil to tripalmitin were recorded during a cool-heat-cool cycle (75 to 5 to 75 to 5 °C) to determine the influence of lipid phase composition (0 or 25% fish oil) on polymorphic transitions and particle stability (Figure 5). The emulsified tripalmitin crystallized at about 21 °C during the first cooling cycle in the absence of fish oil (Figure 5a), whereas it crystallized at about 18.0 °C in the presence of 25% fish oil (Figure 5b). In both emulsions, a single crystallization peak (Tc) was observed, which can be attributed to crystallization of the tripalmitin into the metastable R polymorphic form during cooling. However, it is noticeable that Tc decreased in the presence of fish oil, which is similar to as observed for the bulk oil (Figure 2a). When the 0% fish oil sample was heated, a sharp endothermic peak was observed at about 44 °C corresponding to the R-form melting temperature (Tm,R), then a small exothermic peak was observed around 48 °C corresponding to a melt-mediated transition to the stable β-form, and then a broad endothermic peak was observed at about 62 °C corresponding to the β-form melting temperature (Tm,β). In contrast, when the 25% fish oil sample was heated, a single melting peak was observed at about 59 °C, which corresponds to the melting temperature of the β-form (Tm,β). This result indicated that the presence of this amount of liquid oil within the lipid droplets accelerated the
Figure 5. DSC thermograms of emulsified tripalmitin without (a) and with 25% fish oil (b) during a cool-heat-cool cycle between 5 and 75 °C. The arrows indicate the temperature changes during the cycle.
R-to-β transition of the tripalmitin within the nanoparticles, as was observed for bulk tripalmitin (Figure 3a). When the 0% fish oil sample was recrystallized by cooling it a second time, its thermal behavior was distinctly different from that observed during the first cooling step (Figure 5a): two exothermic peaks (40 and 21 °C) were observed during the second cooling step, whereas only one exothermic peak (21 °C) was observed during the first cooling step. The appearance of an additional exothermic peak at higher temperatures during the second cooling step can be attributed to extensive droplet coalescence during the cool-heat-cool cycle. Droplet coalescence leads to an increase in the mean particle size of the lipid droplets, which means that nucleation occurs predominantly through a heterogeneous mechanism similar to that observed in bulk oil, rather than through a homogeneous mechanism similar to that observed in highly emulsified oils.30-32 Previous studies have shown that extensive particle aggregation often occurs when the crystals in SLN undergo a polymorphic transition from the R- to β-form due to a change in particle shape.13,14 Tripalmitin SLN in the R-form tend to be roughly spherical in shape, whereas those in the β-form tend to be more platelet-like.25,33-35 Consequently, there is an increase in the overall surface area of the particles after an R- to β-polymorphic transition, which means that there may be insufficient emulsifier present to cover the increase in lipid phase surface area. These uncovered surfaces increase the hydrophobic interactions between the particles, which promote flocculation, coalescence and eventually phase separation.13,14 In contrast, when the 25% fish oil sample was recrystallized by cooling it a second time, its thermal behavior was the same as that observed during the first cooling step (Figure 5b), which suggests that the particles were stable against aggregation and phase separation in this system.
Stability of Omega-3 Encapsulated SLN Suspensions
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Figure 7. Plots of (a) crystallization temperature (Tc) and (b) melting temperature (Tm) of emulsified tripalmitin versus percentage of the fish oil (Φ).
Figure 6. DSC melting endotherms (a) and recrystallization exotherms of solid tripalmitin solid lipid nanoparticles containing various concentrations of fish oil during a cool-heat-cool cycle between 75 and 5 °C.
Further insights into the impact of lipid composition on the properties of tripalmitin SLN were obtained by measuring the thermal behavior of emulsions containing varying amounts of fish oil (Figure 6). All samples containing fish oil (10 to 50%) exhibited a single exothermic crystallization peak during cooling (Figure 6b) and a single endothermic melting peak during heating (Figure 6a), with the transition temperatures (Tc and Tm) decreasing with increasing fish oil content (Figure 7). The enthalpy change associated with the transitions (area under the peaks) decreased with increasing fish oil concentration, which can be attributed to the fact that the liquid oil phase dissolves some of the solid fat phase and so there is a small amount of solid fat involved in the transition.36 In addition, the tripalmitin molecules may not be packed as efficiently into the crystals in the systems containing fish oil. Overall, the impact of the fish oil on the phase behavior of the emulsified tripalmitin was qualitatively similarly to that of the bulk tripalmitin (Figures 1 to 3). Effect of Fish Oil on Gelation of Tripalmitin SLN Suspensions. Rheological measurements were performed to monitor the effect of fish oil on the aggregation and gelation of tripalmitin SLN suspensions. We measured the temperature dependence of the storage modulus (G′) and loss modulus (G′′) of the suspensions, and then calculated the complex shear modulus (G* ) (G′2 + G′′2)1/2) and phase angle (δ). The phase angle provides a measure of the fluidity of the sample, varying from 90° for an ideal liquid to 0° for an ideal elastic solid, whereas G* provides a measure of the overall rigidity of the
Figure 8. Temperature-dependent on (a) complex shear modulus (G*) and (b) phase angle (δ) of emulsified tripalmitin without (open squares) and with (open circles) 25% fish oil during cooling from 37 to 5 °C. An additional heating process was done for the sample containing 25% fish oil (closed circles). The arrows indicate cooling and heating, and the numbers refer to the order.
material. An increase in G* or a decrease in δ therefore indicates the formation of a particle network within a SLN suspension. The dependence of the complex shear modulus (G*) and phase angle (δ) of emulsions containing different amounts of fish oil on temperature during cooling and heating was measured (Figure 8). Without fish oil (100% tripalmitin), the complex modulus was close to zero and the phase angle was high (90°) when the emulsions were cooled from around 75 to 16 °C, which
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Figure 9. Photograph of emulsified tripalmitin without fish oil (A), and with 25% (B) or 50% (C) fish oil mixture. Samples were held overnight at 5 °C to induce the SLN formation.
indicated that the emulsions remained fluid over this temperature range. Below 16 °C, the complex modulus rose steeply and the phase angle fell steeply, indicating that the emulsions gelled around this temperature. The rigidity of the gels continued to increase when they were cooled further to 5 °C (Figure 8a). After gelation had occurred, the phase angle of the gels remained low but decreased slightly during cooling to 5 °C (Figure 8b). The increase in gel rigidity upon cooling below 16 °C suggests that the attractive forces between the lipid particles, which crystallized at about 20 °C (Figure 1a), increased due to the development of a particle network. When the gelled SLN suspensions were heated, they remained solid-like up to temperatures where the tripalmitin droplets melted, then they became fluid, and a layer of oil was observed on their top (data not shown). In contrast, emulsions containing 25% fish oil did not show any sign of gelation with the sample remaining fluid during the first cooling process and during subsequent heating and cooling cycles. The addition of relatively small amounts of fish oil was therefore able to prevent extensive particle aggregation in the SLN suspensions. The impact of fish oil on the gelation of the emulsions was also studied by visual observation of the samples after they were quench-cooled to 5 °C to initiate tripalmitin crystallization and then stored for 24 h at this temperature. The sample containing no fish oil formed a strong gel after this procedure, whereas all the samples containing fish oil (g10%) remained fluid and did not show any sign of gelation, even after storage for two weeks
(Figure 9). A clear oil phase was observed on top of the emulsions containing no fish oil after they were heated to 75 °C, indicating that extensive droplet coalescence had occurred. On the other hand, no signs of oiling-off (visual observation) or droplet aggregation (light scattering) were seen when the samples containing fish oil were heated to 75 °C, indicating that they were stable to droplet coalescence. Effect of Fish Oil on Morphology of SLN. To monitor the possible morphological transitions from spherical to needle shaped particles owing to polymorphic transitions,13,14,25 dynamic light scattering was used for samples containing different amounts of fish oil (0 and 25%). Prior to analysis, the emulsions were stored at a temperature where the lipid phase remained liquid (i.e., 37 °C). At this temperature we observed no significant change in their mean particle diameter (147 ( 1 nm) over time, indicating that they were stable to droplet aggregation and Ostwald ripening during storage. The change in hydrodynamic radius with time was then measured when the emulsions were cooled to 15 °C to initiate tripalmitin crystallization (Figure 10). In the absence of fish oil, there was a steep increase in the mean particle diameter of the suspensions, which can be attributed to changes in the particle shape when crystallization and polymorphic transitions (R-to-β) occur. For example, previous studies have shown that pure tripalmitin particles change from a spherical to a needle shape when they crystallize and transform to the β-form,25,33-35 eventually leading to
Stability of Omega-3 Encapsulated SLN Suspensions
Figure 10. Development of the hydrodynamic radius with time for the solid lipid nanoparticles of 100% tripalmitin (open symbols) and those of 75% tripalmitin/25% fish oil mixture (closed symbols) during holding at 5 °C.
extensive particle aggregation and network formation.13,14 In the presence of 25% fish oil, the mean particle diameter of the particles did not change during 600 min storage, indicating that the fish oil prevented the particle shape changes. Conclusions Our results suggest that the addition of a low melting lipid (fish oil) to a high melting lipid (tripalmitin) modified its phase behavior (crystallization, melting, and polymorphic transitions) in a manner that changed the stability of SLN suspensions to particle aggregation and gelation. The DSC data suggests that the polyunsaturated fatty acid chains within the fish oil accelerate the R- to β-polymorphic transformation of the saturated fatty acid chains within the tripalmitin. In addition, DSC showed that the crystallization and melting temperatures of tripalmitin decreased with increasing fish oil content, which can be attributed to the formation of less ordered crystals. The tendency to form less ordered crystals in the presence of liquid oil may account for the fact that the fish oil inhibited droplet shape changes during crystallization and polymorphic transitions of the emulsified tripalmitin, thereby inhibiting particle aggregation and network formation. Our results show that fish oil, rich in ω-3 polyunsaturated fatty acids, can be successfully incorporated into SLN suspensions. In future studies, we intend to examine the impact of incorporation of fish oils into tripalmitin nanoparticles on their susceptibility to lipid oxidation. Potentially, SLN could be used to stabilize fish oils against oxidation by retarding molecular diffusion and keeping polyunsaturated lipids isolated from pro-oxidants. Acknowledgment. This grant was supported by USDA CSREES Hatch grants (MAS 0911 and MAS 831) and grants by the USDA National Research Initiative Programs (Award Number 2005-01357). We thank Professor Shaw Ling Hsu and Dr. Huimin Bao of the Polymer Science and Engineering Department at University of Massachusetts for using of and assistance with the NMR spectroscopy. We also thank Dr. Dalal Asker for critical discussions and for the TOC graphic design. Note Added after ASAP Publication. After this paper was published ASAP June 19, 2009, a correction was made to the third paragraph under “Effect of Fish Oil on Crystallization of Emulsified Tripalmitin”; the corrected version was reposted June 29, 2009.
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